Our research goals are: (1) to determine growth requirements for serial and long-term cultivation of normal tracheal epithelial cells in vitro, (2) to investigate series of events occuring on differentiative features (cilia and mucus granules) of tracheal epithelial cells after plating in culture, (3) to elucidate differentiative properties of cultured tracheal cells and their differentiative potential. In vitro culture conditions have been developed to permit serial and clonal cultures of tracheal epithelial cells from rabbits, rats, hamsters and mice. These cells exhibited 10 to 30 population doublings of in vitro life span, and confluent cultures could be passaged 3 to 5 times. We have observed a rapid loss of cilia and mucus granules of cells in culture. Except hamster cells, new cilia were formed after the confluency of culture. Repopulation studies have shown that cultured cells regained mucociliary functions after plating in denuded tracheal grafts. Furthermore, all cultured cells maintained the synthesis and secretion of mucin-like glycoprotein and they were also able to express squamous epithelial properties, which included stratification, cornification and keratinization. These results suggest that these in vitro culture systems can be used to study cell differentiation of tracheal epithelial cells.